The majority of severe vision loss in the US results from complications associated with retinal neovascularization in patients with ocular diseases, including diabetic retinopathy (DR) and Age- Related Macular Degeneration (AMD). These diseases are characterized by excessive angiogenesis, which promotes vascular leak leading to edema, hemorrhage and retinal detachment compromising vision. To date, the treatment of excessive angiogenesis relies largely on inhibition of a single factor, vascular endothelial growth factor (VEGF), with some therapeutic success. Clinical trials have shown that anti-VEGF treatment reduces angiogenesis in AMD and DR patients, indicating that anti-angiogenic treatment is a viable therapeutic option. However, treatments involve frequent intraocular injections, and a proportion of patients do not achieve vision improvement. Therefore, there is enormous therapeutic interest to develop treatments that enhance response to anti-VEGF agents and improve the lives of patients with ocular neovascular disease. We here identify the guidance molecule Slit2 as a requisite factor that promotes angiogenesis in addition to VEGF. Disruption of Slit2 function by temporally inducible deletion of the Slit2 gene, and combined deletion of its Robo1 and Robo2 receptors in ECs potently inhibits retinal neovascularization. These data lead us to test the hypothesis that blocking Slit2 signaling through Robo1 and 2 represents an alternative pathway to block excessive angiogenesis that may serve as a therapeutic target in conjunction with anti-VEGF treatment of AMD. Mechanistically, we will test the hypothesis that blocking of Slit2-Robo1/2 signaling improves efficacy of anti-VEGF treatment by selectively targeting EC migration and define the signaling pathways regulating this process. Our studies will provide the first comprehensive examination of Slit-Robo1/2-mediated angiogenesis in ocular neovascularization, determine its biological significance and define the molecular and cellular basis of Slit signaling through Robo1/2 signaling. Understanding these mechanisms will provide insight into signaling events required for polarized endothelial cell migration during neovascularization, which is a fundamental, yet poorly understood event in vascular biology. The ultimate goal of our proposal is to develop new therapies to prevent intraocular vascular disease.